The role of TPA and/or cholera toxin on the melanocyte development in Mitf^<mi-ew> mouse

已经证明Mitf不仅是酪氨酸酶、Tyrp 1和Tyrp 2的转录因子，而且是黑素细胞的存活因子。为了研究Mitf在黑素细胞存活中的作用，我们进行了神经嵴细胞（NCC）原代培养，其中将来自Mitf+小鼠的神经管外植体<mi-ew>在15%FBS/Eagle MEM中培养。在这些培养物中，即使当向培养物中加入Kitl和/或Edn 3时，也没有出现Kit、酪氨酸酶、Tyrp 1或Tyrp 2阳性的细胞。当用TPA和霍乱毒素培养时，出现了一些Tyrp 1阳性细胞和较少数量的Kit阳性细胞，但所有细胞中的酪氨酸酶和Tyrp 2仍为阴性。为了阐明Mitf在维持黑素细胞中的机制，我们通过RNA干扰技术敲低来自小鼠NCC的Kit和DOPA阴性的未成熟黑素细胞系NCCmelb 4 M5中Mitf的表达。添加Mitf-M的siRNA抑制细胞增殖，然后引起细胞从培养瓶中脱离。免疫组化显示caspase 3阳性细胞呈时间依赖性增加，提示细胞凋亡诱导。Real-time PCR分析显示，Mitf-M siRNA的加入抑制了Tyrp 1 mRNA的表达，增强了bax mRNA的表达。由于bcl-2的表达没有改变，可能是bax/bcl-2比例失衡导致细胞凋亡。当TPA和霍乱毒素在加入Mitf-M的siRNA之前不久加入时，细胞免于细胞死亡。在这些细胞中，Tyrp 1和bcl-2的mRNA表达上调，而bax没有变化。目前的研究结果表明，Mitf通过Tyr的诱导在早期发育阶段的黑素细胞存活中发挥作用。

It has been demonstrated that Mitf is not only a transcription factor for tyrosinase, Tyrp1 and Tyrp2 but also a survival factor for melanocytes. To investigate the role of Mitf in melanocyte survival, we performed neural crest cell(NCC) primary cultures in which explants of neural tubes from Mitf^<mi-ew> mice were cultured in 15% FBS/Eagle MEM. In these cultures, cells positive for either Kit, tyrosinase, Tyrp1 or Tyrp2 did not appear, even when Kitl and/or Edn3 were added to the cultures. When they were cultured with TPA and cholera toxin, some Tyrp1-positive cells and fewer numbers of Kit-positive cells appeared, but tyrosinase and Tyrp2 were still negative in all cells. To clarify the mechanisms of Mitf in maintaining melanocytes, we knocked down the expression of Mitf in NCCmelb4M5,which is a Kit-and DOPA-negative immature melanocyte cell line derived from mouse NCC, by the RNA interference technique. Addition of siRNA of Mitf-M inhibited cell proliferation, and then caused detachment of the cells from the culture flasks. Immunostaining showed that caspase 3-positive cells increased time-dependently, suggesting the induction of apoptosis. Real-time PCR analysis revealed that the addition of siRNA of Mitf-M inhibited Tyrp1 mRNA expression and enhanced bax mRNA expression. Since the expression of bcl-2 was not altered, it is likely that the imbalance of bax/bcl-2 ratio led to apoptosis. When TPA and cholera toxin were added shortly before the addition of siRNA of Mitf-M, the cells were rescued from cell death. In these cells, the mRNA expression of Tyrp1 and bcl-2 were up-regulated, while bax did not change. The present findings suggest that Mitf play a role in melanocyte survival in early developmental stages through the induction of Tyr